Copyright ©Environmental Fredy A Rivera Páez CRIMSON PUBLISHERS C Wings to the Research Analysis & Ecology Studies ISSN 2578-0336 Review Article Genus 1 sp. 2 (Diptera: Chironomidae): The Potential use of its Larvae as Bioindicators Paula A Ossa López1, Narcís Prat2, Gabriel J Castaño Villa3, Erika M Ospina Pérez1, Ghennie T Rodriguez Rey4 and Fredy A Rivera Páez1* 1Grupo de Investigación GEBIOME, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Colombia 2Grup de recerca consolidat F.E.M (Freshwater Ecology and Management), Departament de Biologia Evolutiva, Ecologia i Medi Ambient, Universitat de Barcelona, Barcelona, España 3Grupo de Investigación GEBIOME, Departamento de Desarrollo Rural y Recursos Naturales, Facultad de Ciencias Agropecuarias, Universidad de Caldas, Manizales, Caldas, Colombia 4Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Caldas, Colombia *Corresponding author: Fredy A Rivera Páez, Grupo de Investigación GEBIOME, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Caldas, Manizales, Caldas, Colombia Submission: November 05, 2018; Published: November 16, 2018 Abstract The family Chironomidae belongs to the most abundant macroinvertebrates in samples for water quality assessment and displays a wide tolerance range to contaminants, which makes it an excellent bioindicator. The species Genus 1 sp. 2 (Chironomidae: Orthocladiinae), included among the larval Chinchinákeys of the river Cricotopus-Oliveiriella basin (Caldas-Colombia), complex, along is difficultwith eight to organismsdetermine atbased the pupal on its level larval (reared instar in using the laboratory). the current The morphological organisms were keys, morphologically which makes it necessary to use pupae for a species-level identification. In this study, 103 organisms in the IV larval instar were collected from tributaries of the high identified, and a molecular analysis of the genes COI and 16S rDNA was performed in order to confirm and associate larvae and pupae. In the larval morphometric analysis, 13 structure measurements were taken, with the aim of finding possible variations among specimens from samplingdifferent samplingstations. Thestations, results and obtained only dorsal allow head for a areamolecular (DHAr) determination showed significant and association differences. of larvaeThe presence and pupae of mentumof the species deformities Genus 1was sp. assessed,2, and new a morphologicaltotal of 18 specimens measurements showed inpartial larvae or that total can teeth aid indeformity, determining although variations no significant resulting differencesfrom contaminant were found agents between and contributing deformity tofrequency establishing and thisthe species as a water quality bioindicator. Keywords: Colombia; Deformities; Molecular analysis; Morphology; Morphometry Introduction The subfamily Orthocladiinae (Diptera: Chironomidae) is one of species-level, the differences between species have not been studied the richest in genera and species, and in Andean rivers above 2000 complicating its identification even at the genus level, and at the yet. Its pupae, however, are very characteristic and very different meters of altitude, the subfamily Orthocladiinae is very abundant, from the genus Cricotopus; therefore, a species-level description can with multiple genera present in the high Andean region, and of be achieved. Larvae belonging to this taxon are abundantly found in which some have yet to be described [1]. Furthermore, species of the Chinchiná River (Colombia), and based on studies related to the the same genus share many larval characteristics, making it nearly association between macroinvertebrates and contamination, the impossible to distinguish them, even at a genus level. Nevertheless, possibility of using these larvae as contamination indicators has led to a complete morphological and genetic study in order to establish there are several records in which larvae and pupae have been pupal forms are specific for a genus, and even for a species, and its possible use as bioindicators [4,5]. associated in rivers of the high Andean region between Colombia and Peru. The use of aquatic macroinvertebrates currently constitutes a tool for the biological and integral characterization of water Among the most abundant genera of Orthocladiinae in the high quality [4,6]. All aquatic organisms can be considered as Andean region, there are larvae described as Genus 1 by Roback bioindicators, however, the evolutionary adaptations to different & Coffman [2], a genus found exclusively in the Andean region. environmental conditions and the tolerance limits to a given Its larvae belong to the Cricotopus-Oliveiriella complex [1,3]; disturbance are responsible for the characteristics that classify Volume -4 Issue - 3 Copyright © All rights are reserved by Fredy A Rivera Páez. 376 Environ Anal Eco stud Copyright © Fredy A Rivera Páez them as sensitive organisms, whether they do not endure changes in their environment or they are tolerant to stress conditions. of 250µm, and manual drainers (the samples were taken from a Surber net, with 30.5x30.5x8cm dimensions and a mesh size Chironomidae (Diptera: Chironomidae), with nearly 20000 species sediments, rock washes and leaf litter). The specimens were distributed throughout all the continents, from the Antarctic region preserved in absolute ethanol with their corresponding information to the Tropics, inhabit lakes, streams and rivers during their larval (date, location, and coordinates). In the laboratory, several and pupal developmental stages [1,2,4]. specimens were conditioned in aquariums with water from the sampling stations, under constant oxygenation, and were fed with The family Chironomidae is considered to be tolerant to water TetraMin® contamination with organic matter, heavy metals, pesticides, aromatic polycyclic hydrocarbons, and organic solvents, displaying Additionally, until pupaethe werefollowing obtained physical for species and confirmation.hydrobiological subletal responses such as morphological variations represented variables were measured in situ in three of the six sampling events: by morphometric changes and deformities as a result of exposure to water temperature, pH, conductivity, dissolved oxygen, oxygen these conditions [5-11]. Regarding these tolerance characteristics, the following chemical variables were evaluated in the laboratory: saturation, average depth, width, and water flow velocity. Also, et al. [14,15], Giacometti & Bersosa [16], report Chironomidae chemical oxygen demand (COD), biological oxygen demand (BOD ), Arambourou et al. [11], Warwick [12], Alba-Tercedor [13], Servia 5 as organisms with a potential use in water quality bioindication. Nevertheless, one of the current limitations for the use of total coliforms, fecal coliforms, total suspended solids (TSS), total ammoniacal nitrogen (NH -N), phosphate (PO ), solids (TS), cyanide (CN), 3boron (B), lead (Pb),4 mercury (Hg),4 which in many cases is not straightforward, due to phenotypic iron (Fe), chloride (Cl-), lipids and oils, nitrates (NO ), nitrites Chironomidae is an insufficient knowledge of their taxonomy, ), sulfate3 (SO plasticity or shared characters between several species of a genus, (NO2), and aluminum (Al). These variables were analyzed by or even between genera. chemical variables between the reference and mining stations were The present study aimed to morphological evaluation the ACUATEST S.A. Comparisons of the physical, hydrobiological, and performed using the Wilcoxon test (W). larvae of Genus 1 sp. 2 Roback & Coffman [2], through diagnostic characters, further, larvae and pupae of Genus 1 sp. 2 were Morphological and molecular evaluation molecularly determined and associated based on the study of The ethanol-preserved organisms were examined and mitochondrial genes. The possible morphometric variations and record the frequency of deformities was assessed in organisms of the stereomicroscope equipped with a MC170HD digital camera. Next, identified based on the keys of Prat et al. [1,3], using a Leica M205C the head of each specimen, both the larvae and pupae reared in impact or lack of any evident mining impact and sampling stations IV larval instar in the sampling stations (no evident anthropogenic the laboratory, were dissected and placed in hot 10% KOH. They with mining impact). Overall, the results allowing to contribute to were then washed, dehydrated, and mounted on microscope slides the establishment of this species as a water quality bioindicator. with Euparal® for their subsequent observation, following the light Materials and Methods microscopy (LM) techniques described by Epler [19], and the head capsule and pupal keys of Prat et al. [1,20]. Study area DNA was extracted from the thorax and abdomen of 11 larvae, The study area included six sampling stations located in the as well as two mature pupae, using the DNeasy Blood and Tissue Chinchiná River basin in the department of Caldas (Colombia). Kit (Qiagen®), according to the manufacturer’s instructions. Two sampling stations were selected as reference (areas without Two mitochondrial genes (mtDNA), cytochrome oxidase I (COI) an evident mining impact), one located in La Elvira stream (05°03’10.9’’ North, 75°24’33.6’’ West), municipality of Manizales, that were conducted following Ossa et al. [21]. The PCR products and the other in Romerales stream